Oral Toxicity and Bacterial Mutagenicity Studies with a Spunbond Polyethylene and Polyethylene Terephthalate Polymer Fabric

Abstract

Spunbond, nonwoven fabrics consisting of polyethylene and polyethylene terephthalate, which meet food contact requirements, may be used as pouch materials for products containing food and/or flavor ingredients that are held in the mouth. In these situations ingestion may occur, resulting in exposure to the fabric and potentially antimony, a catalyst used in polyethylene terephthalate. To assess potential adverse effects when such a material is ingested, a 13week dietary study in Sprague-Dawley CD rats and a Salmonella reverse mutation assay were conducted. Ground fabric was dosed at target concentrations of 0.5%, 2.5%, and 5% in the dietary study. Antimony trioxide in the polyethylene terephthalate was used to determine the test material concentration in the diet and was also assessed for bioavailability. Detectable levels of antimony were found in 2/20 blood samples of control rats, and in 20/20 high-dose group rats. No toxicologically relevant treatment related effects were observed in any of end points evaluated in the feeding study. In the mutagenicity assay, ground fabric was extracted in phosphate buffered saline or dimethysulfoxide and tested in Salmonella strains TA98, TA100, TA102, TA1535, and TA1537 with and without S9 activation. No mutagenic response was observed at any dose level tested. These results demonstrate that repeated daily ingestion of a spunbond, nonwoven polymer fabric consisting of polyethylene and polyethylene terephthalate for up to 13 weeks is well tolerated in rats, with no apparent target-organ toxicity at dietary levels up to 5%, and that fabric extracts are not mutagenic in a bacterial reverse mutation assay.

Spunbond, nonwoven polymer fabrics made of polyethylene (PE) and polyethylene terephthalate (PET), which meet the requirements under the United States Food and Drug Administration (FDA) regulations 21CFR 177.1630 and 177.1520, may be used in food contact applications. Although typically not ingested under these traditional conditions of use, such PE/PET polymer fabrics may also find applications in oral pouch products containing various food, flavoring, and other ingredients. In such applications, the pouch is placed in the mouth for short periods of time, during which soluble components are extracted. Although ingestion of these products during use in not intended, it is not preventable.

Ingestion of PET may result in potential exposure to antimony, because antimony trioxide is commonly used as an initiator in the production of PET (ILSI 2000; Westerhoff et al. 2008; WHO 2003). PET is widely used to make drinking water bottles and has, under some conditions, been shown to leach antimony into drinking water (Shotyk and Krachler 2007; Westerhoff et al. 2008).

Although a variety of safety information is available on PE and PET (CIR 2007; ILSI 2000), relatively little information was found to assess the potential toxicity of these materials in situations where unintended ingestion occurs. In addition, no studies were found in which PE and PET were administered in combination, or which examined the potential bioavailability of antimony from PET. To help address this information gap, a PE/PET polymer fabric was evaluated in a 13-week feeding study and a Salmonella reverse mutation assay. These studies are recognized as standard methods for toxicity evaluations (FDA 2000) and were conducted according to Organization for Economic Cooperation and Development (OECD) Guidelines (OECD 1998; OECD 1997), and Food and Drug Administration (FDA) Good Laboratory Practice Regulations 21CFR Part 58. In the feeding study, antimony was used as a marker for determining the level of the test material in the diet. In addition, analytical studies were conducted to determine if the antimony in the PET was bioavailable.

MATERIALS AND METHODS

Test Material and Preparation

The test material (Ahlstrom 25707), a nonwoven, spunbond polymer fabric made of PE (CAS no. 2622-73-8) and PET (CAS no. 25038-59-9), was obtained from Ahlstrom Nonwovens (Windsor Locks, CT). Both the PE and PET used in the fabric were certified by the supplier to be in accordance with FDA regulations 21CFR 177.1630 and 21CFR 177.1520, substances for use as basic components of single and repeated use food contact surfaces.

To facilitate inclusion in the test diet, the test material was milled into a fine powder. To do this, the PE/PET polymer fabric was cut using scissors into small pieces about 1 cm², which were then placed into a grinding vial of a 6850 Freezer/Mill (SPEX SamplePrep, Metuchen, NJ) and milled. The milling procedure consisted of chilling the vial in liquid nitrogen for 15 min, then running the mill for five 2-min grinding cycles. Each grinding cycle was followed by 2 min of recooling. The dimensions of the particles obtained after the grinding procedure were not determined, but a prestudy palatability test confirmed that the ground material would be ingested by the test animals.

13-Week Feeding Study

Test Animals

This study was conducted in compliance with all appropriate parts of the Animal Welfare Act regulations (9 CFR Parts 1 and 2 Final Rules). Male and female Sprague-Dawley [Crl:CD(SD)IGS BR] rats were obtained from Charles River Laboratories (Portage, MI). The rats were approximately 6 weeks of age upon arrival and were held in quarantine for 1 week prior to study initiation. Animals were housed in accordance with the Guide for Care and Use of Laboratory Animals (NRC 1996) using stainless steel cages equipped with a feeder and an automatic watering system which provided City of Chicago water ad libitum. Upon receipt, rats were housed up to two per cage to facilitate acclimation to the automatic watering system. Before treatment began, and throughout the remainder of the study, rats were housed individually. Absorbent cage boards were placed underneath the cages to retain liquid and solid waste. Room temperature was maintained at 19°C to 25°C and the humidity at 40% to 70%. Group assignment occurred one day prior to study initiation using a body-weight randomization program. At initiation the body weights were within 20% of the mean weight for the group for the respective sex.

Test Procedure

This study was conducted in accordance with the OECD Guideline for Testing Chemicals (OECD 1998). The test material was administered orally via the diet for 13 consecutive weeks to 10 male and 10 female rats per group. Feed consumption and body weights were recorded weekly. Prior to group assignment, the eyes of all animals received were examined using an indirect ophthalmoscope. The eyes of all surviving animals were reexamined during week 13. Cage-side clinical observations were performed daily. Detailed clinical observations, including activity levels and locomotion, skin and coat condition, eye and mucous membrane condition, any altered behavior or other relevant observations were performed weekly. Hematology, coagulation, and clinical chemistry were performed on surviving animals prior to study termination. Complete necropsies were conducted and selected organs were weighed. Microscopic examination of selected tissues was conducted on the control and 5% dose group animals.

Preparation and Administration of Test and Control Diets

Certified Purina Rodent Chow/Meal 5002 (PMI Nutrition International, Brentwood, MO), was used to prepare the test diets. Control animals received unsupplemented diet.

The test diet was prepared by mixing the ground test material in the basal diet at target levels of 0.5%, 2.5%, and 5% using a V-blender (Blend Master Lab Blender Model B, Patterson-Kelly, East Stroudsburg, PA). The 5% concentration tested was targeted to avoid introducing adverse nutritional effects and to adequately exceed anticipated human intake. Test diets were prepared prior to study initiation and about every 1 to 2 weeks during the study. An analytical method for determining the level of the test material in the dietary admixtures was developed and validated using antimony as the marker. The first four batches of the diet prepared, a batch in the middle of the study as well as the last batch of diet prepared were analyzed for concentration of the test material. Homogeneity of the test material in the diet was determined on the first batch of diet prepared, a batch prepared in the middle of the study and the last batch prepared. Samples were collected from three different areas of the diet mixture (left, right, and bottom portions of the blender). For stability analysis, samples were collected from the first batch and an aliquot was analyzed prior to use. Aliquots of the remaining sample were stored at room temperature and reanalyzed weekly for five weeks. The test diets and control diet were administered ad libitum for at least 13 weeks.

In-Life Parameters

Cage-side observations of all animals for mortality, moribundity and general appearance were made at least twice per day. Detailed hand-held clinical/physical observations were made on all animals weekly throughout the study. These observations included, but were not limited to, the following: activity levels and locomotion; skin or coat condition; eyes and mucous membranes condition; the respiratory, circulatory, and nervous systems; altered behavior; unusual discharge of body fluids; and other relevant observations. Feed consumption was measured individually for each animal concurrently with body weight measurement at weekly intervals during the study.

Hematology, including coagulation, and clinical chemistry studies were performed on all surviving animals at termination of the study. Blood samples for hematology and clinical chemistry were obtained from the retro-orbital plexus of animals anesthetized with 70% CO₂/30%air, whereas blood samples for coagulation parameters were obtained from the abdominal aorta. Animals were fasted overnight prior to blood collection.

The following hematology end points were recorded on the day of collection using the ADVIA 120 Hematology System (Siemens Medical Solutions Diagnostics, Tarrytown, NY): erythrocyte count, erythrocyte morphology, absolute white blood cell count, relative white blood cell count, hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, and platelet count.

Blood for clinical chemistry studies was collected into tubes, allowed to clot, centrifuged to obtain serum, and assayed on the day of collection. The automated microanalysis for the following were performed using a Synchron LX20 Analyzer (Beckman Coulter, Fullerton, CA): A/G ratio (derived), albumin (A), alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total), blood urea nitrogen, calcium chloride, cholesterol, creatinine, γ-glutamyl transpeptidase, glucose, globulin (G, total, derived), lactate dehydrogenase, inorganic phosphate, potassium, sodium, total protein, and triglycerides.

Blood for coagulation studies was collected into tubes containing citrate anticoagulant. The following parameters were measured using a STA Compact CT coagulation analyzer (Diagnostica Stago, Parsippany, NJ): prothrombin time, fibrinogen, and activated partial thromboplastin time.

Blood and Fecal Antimony Analysis

Whole-blood (retro-orbital sinus collection with ethylenediaminetetraacetic acid anticoagulant), and feces samples from the control and high-dose test groups were obtained 1 day prior to necropsy and analyzed for the presence of antimony. Approximately 0.5 ml of blood and 0.1 g of feces were digested with concentrated nitric and hydrochloric acids in a Multiwave 3000 Microwave Sample Preparation System (Anton Paar USA, Ashland, VA). After digestion, the samples were diluted to 30 ml with distilled water and analyzed directly on a Perkin Elmer Elan DRC II ICP-MS (PerkinElmer Instruments, Shelton, CT) equipped with a dynamic reaction cell and PerkinElmer AS-93 Plus autosampler.

Postmortem Parameters

Surviving animals were necropsied after a minimum of 91 days of treatment. Necropsies were performed in a predesignated order across groups by sex. Animals were euthanized by exsanguination under sodium pentobarbital anesthetic. Overnight-fasted body weights were collected at necropsy. Necropsy included examination of the external surface of the body, all orifices, the cranial, thoracic, and abdominal cavities and their contents. The spleen, adrenals, thyroid (with parathyroids), thymus, testes, ovaries, kidneys, heart, liver, and brain were taken from all surviving animals, weighed, and organ/body weight ratios calculated using the final overnight-fasted body weights. To prevent possible tissue damage associated with weighing, the thyroid and parathyroids were weighed after 24 h of formalin fixation.

The tissues listed in Table 1 were obtained and preserved from all animals. All tissues, except eyes, harderian glands, and testes were preserved in 10% neutral-buffered formalin. Testes were preserved in Bouin’s fixative and eyes and harderian glands in Davidson’s fixative. Lungs and urinary bladder were infused with formalin to ensure fixation.

Histopathological evaluation of the tissues listed in Table 1 from all rats in the control group (group 1) and in the high-dose group (group 4) was conducted by a board-certified veterinary pathologist. Tissues from the low- and mid-dose groups were embedded in paraffin blocks. Histopathological evaluations of tissues from rats in the low- and mid-dose groups were limited to gross lesions and identified target tissues. Tissues to undergo histopathological evaluation were trimmed, processed, sectioned, and then stained with hematoxylin and eosin.

Statistical Evaluations

Clinical observations were tabulated, but not statistically analyzed. Body weight, food consumption, hematology, coagulation, clinical chemistry, and organ weights (absolute and relative) data were analyzed by analysis of variance followed, where appropriate, by the post hoc Dunnett’s test. LABCAT recorded data (body weight, weight gain, and clinical pathology data) were analyzed using LABCAT statistical software (version 4.65 [body weight and gain] and version 4.43 (clinical pathology); Innovative Programming Associates, Princeton, NJ). All analyses were performed for two sexes independently. A significance level of p ≤ .05 was used in all comparisons.

Salmonella Reverse Mutation Assay

The mutagenic activity of the phosphate-buffered saline (PBS) and dimethylsulfoxide (DMSO) extracts of the synthetic non-woven material were determined in five different Salmonella typhimurium tester strains (TA98, TA100, TA102, TA1535, and TA1537) with and without exogenous S9 metabolic activation. Both a dose range–finding and a confirmatory assay were performed.

Test Material Extraction

One day before the assay, 1-g samples of the ground test material were weighed, placed in separate borosilicate glass tubes (with screw cap), and extracted either with 10 ml of PBS without Ca²⁺ or Mg²⁺ (Biowhittaker, Walkersville, MD) or 10 ml of DMSO (Sigma-Aldrich, Milwaukee, WI), respectively, in a shaker incubator with shaking at approximately 300 rpm at 37°C ± 1°C for 24 ± 1 h. Each extract (aqueous or organic) was tested at concentrations of 100% (neat), 50%, and 10% in the dose range–finding mutagenicity and bacterial-cytotoxicity assays. Bacterial cytotoxicity was negative at these concentrations.

Experimental Design and Procedures

The Salmonella reverse mutation assay was conducted in accordance with the methods described by Maron and Ames (1983) and in compliance with OECD Guideline 471 (OECD 1997). Five different bacterial strains were used in this assay (TA98, TA1535, TA1537: Molecular Toxicology, Boone, NC; TA100: Xenometrix, San Diego, CA; TA102: Litron Laboratories, Rochester, NY). The standard plate incorporation method was used to plate bacteria along with the test substance, and the S9 metabolic activation mixture or buffer. For metabolic activation of promutagens, Aroclor 1254–induced rat liver postmitochondrial S9 fraction (Molecular Toxicology) was used. The S9 mixture was filter-sterilized by 0.45-μm syringe filter prior to use, and kept in an ice bath during the experiment.

Bacteria were maintained as stock cultures in Nutrient Broth No. 2 (OXOID, Hampshire, England) with DMSO in liquid nitrogen. The broth contained 25 μg/ml ampicillin for TA98 and TA100, and 25 μg/ml ampicillin plus 2 μg/ml tetracycline for TA102. One day before the assay, the tester strain bacteria were cultivated in 20 ml of nutrient broth with approximately 1.0 ml of the thawed stock culture in duplicate. The bacteria were incubated overnight at 37°C ± 1°C to obtain logarithmic/early stationary growth phase cultures.

On the day of the assay, 100 μl of the test material extract, 100 μl bacterial culture, and 500 μl S9 mixture, or 500 μl sodium phosphate buffer for assays without S9, were added to 2 ml of top agar. To cover a dose range the extracts were tested either as neat (100%), 50%, and 10% dilutions. The extract vehicle (PBS or DMSO) served as the control. The components were vortexed and spread evenly on minimal glucose agar plates. After the top agar solidified, the plates were inverted and incubated at 37°C ± 1°C for 48 to 72 h. The number of revertant colonies in each plate was counted in an AccuCount 1000 Automatic Colony Counter (BioLogics, Manassas, VA).

For each bacterial strain and activation condition (with or without S9), three plates for each of the vehicle/solvent control, positive control, and test article were prepared. In addition to the positive and vehicle controls, sterility controls were run simultaneously with the test article.

Sensitivity of the test organisms to the following positive control mutagens was confirmed: 2-aminoanthracene, 2-aminofluorene, cumene hydroperoxide, and acridine mutagen ICR-191 (Sigma-Aldrich, St. Louis, MO); 1,8-dihydroxyhydroquinone, methyl methanesulfonate, and sodium azide (Fisher-Acros Pittsburgh, PA); daunomycin (Molecular Toxicology).

RESULTS

13-Week Dietary Study

Diet Mixture Analysis

Prepared diets were found to be homogenous and stable up to at least 5 weeks. Analysis indicated the diets were within 15% of target concentration, with mean levels of 0.47%, 2.4%, and 4.7% for the low-, mid-, and high-dose groups, respectively.

Mortality and Clinical Observations

There were no deaths during the study. A variety of sporadic clinical observations were found during the study including redness around the eyes, alopecia, broken/misaligned teeth, and lacrimation. All of the clinical observations were considered to be incidental and unrelated to treatment with the test material.

Body Weights, Food Consumption

No statistically significant differences were observed in mean body weights for male or female rats during the study (Figure 1). Weekly body weight values for males and females increased steadily throughout the exposure period. A statistically significant decrease in body weight gain occurred at the 71-day observation period for the mid-dose male rats (data not shown). This was not observed at subsequent time points and was therefore considered incidental. No other statistically significant differences were observed in body weight gain.

Mean weekly feed consumption values for the female high-dose group were significantly greater on days 8 to 57 compared to controls (Figure 2). A similar trend was noted for male rats in the high-dose group compared to control, but the differences between the groups were not statistically significant. These increases were presumed to be related to caloric deficiency due to 5% of the diet being replaced with the polymer material during a period of rapid growth, and were therefore considered to be secondary effects unrelated to test article toxicity.

Hematology, Serum Chemistry, and Coagulation

At terminal necropsy, statistically significant increases in red blood cell count, hemoglobin and hematocrit were observed in male rats in the low-dose group and in females in the low- and mid-dose groups (Table 2). Statistically significant decreases occurred in absolute and relative reticulocyte levels in females in the mid-dose group (Table 2). No treatment-related effects were seen on red blood cell morphology.

No statistically significant differences were noted in any serum chemistry parameters evaluated in male or female rats at terminal necropsy (data not shown).

Statistically significant decreases in fibrinogen levels were seen in males in the low- and mid-dose groups (Table 2). No statistically significant differences were seen in any coagulation parameters in females.

Analysis for Antimony in Blood and Feces

In the control group, levels of antimony in blood were below the limit of quantitation (1.4 ng/ml) for all animals except two where levels were 2.5 ng/ml or less. Antimony was detected in the blood of all 20 high-dose group animals (Table 3). Values ranged from about 12 to about 43 ng/ml (averaging about 20 ng/ml). The increases in the high-dose group values were statistically significant versus the control group whether the “below the limit of quantitation” value was considered to be zero or at the limit of quantitation. Low levels of antimony were present in fecal samples from all animals in the control group, ranging from about 0.1 to 0.2 μg/g (averaging about 0.1 μg/g). In fecal samples from the high-dose group, levels were about 150 times higher and ranged from about 11 to 19 μg/g (average of about 15 μg/g). No substantial differences were noted between the males or females for the presence of antimony in the blood or feces.

Ophthalmic Findings

Ophthalmic examinations were performed before the study and during week 13. No evidence of any ocular toxicity was found in any animal at the end of the treatment period.

Organ Weights

At terminal necropsy, statistically significant increases were present in absolute adrenal, ovary, and spleen weights in the high-dose female group (Table 4). However, relative adrenal and spleen weights were not increased in the high-dose females, so the increased absolute weights were not considered related to test article administration. Relative ovary weights were statistically significantly increased in the high-dose female group (Table 4). However, no microscopic changes were seen in the ovaries of the high-dose females (data not shown) that might be responsible for the increased weight, so this effect was not considered to be caused by the test material. Organ weight measurements for the other organs examined did not suggest any changes indicative of a treatment-related effect.

Necropsy and Histopathological Findings

Gross necropsy findings consisted of the following: one small epididymis; one foot deformity; six enlarged and five pigmented lymph nodes; one enlarged and one small testis; 19 pigmented thymus glands; and two dilated uterine horns. None of these observations appeared to be related to either test material or dose and were interpreted as incidental findings.

There were no microscopic findings in any of the tissues examined that were considered to be related to the administration of the test polymer material. All microscopic findings (data not shown) were consistent with normal background lesions in clinically normal rats of the age and strain used in this study.

Salmonella Reverse Mutation Assay

In the dose range–finding mutagenicity assays, most of the PBS and DMSO extracts of the test article did not demonstrate any indication of a dose-dependent mutagenic response. However, a partial dose-response, i.e., increased responses at the 10% and 100% concentrations, but not the 50%, was seen for the PBS extract in strain TA98 without S9. However, when repeated in the confirmatory mutagenicity assays, none of the PBS or DMSO extracts of the test material showed any dose-dependent mutagenic response (Table 5). In addition, neither the PBS nor the DMSO extracts exhibited bacterial cytotoxicity at any dose level in any strain in the presence or absence of S9 activation.

DISCUSSION

PE and PET are both used in medical device and food contact applications (Brun et al. 1992; ILSI 2000). A review of published toxicological information on PE showed that it was evaluated in a wide variety of safety studies; however, only one subchronic oral study was reported (CIREP 2007). In that study, PE was fed to rats at levels up to 5% in diet for 90 days. No adverse effects were noted.

Information on PET is somewhat more limited than that on PE. A review by the International Life Sciences Institute (ILSI 2000) that focused on food packaging applications stated that PET had been evaluated in animal feeding studies; however, no details on such studies were provided.

PET is a potential source of exposure to antimony trioxide. In a toxicological review of antimony trioxide, the National Toxicology Program noted that various forms of antimony are present in air, soil, and water and that the estimated average daily intake from food and water was 100 μg/day (NTP 2005). Toxicological effects in humans, such as gastritis, respiratory tract irritation, changes in electrocardiograms, and dermatitis have been reported from heavy exposure to antimony compounds, primarily as a result of occupational exposure or their use as therapeutic agents (NTP 2005). It was also noted that antimony trioxide is absorbed following inhalation exposure, but gastrointestinal absorption in man is poor. Based on the results of inhalation studies, the International Agency for Research on Cancer (IARC) classified antimony trioxide as “possibly carcinogenic to humans,” group 2B, i.e., there was inadequate evidence in humans, but sufficient evidence for carcinogenicity in experimental animals (IARC 2008). However, it should be noted that the IARC assessment was done prior to the publication of a study that failed to show a carcinogenic effect in Fischer 344 rats (NRC 2000). In its evaluation of antimony in drinking water, the WHO (2003) notes that although “there is some evidence for the carcinogenicity of certain antimony compounds by inhalation, there are no data to indicate carcinogenicity by the oral route.” In addition, the carcinogenicity of antimony trioxide by inhalation was questioned by Hext, Pinto, and Rimmel (1999), noting that tumors in animal studies likely resulted from overloading of the lung with an insoluble, inert material.

In the present feeding study, Sprague-Dawley rats were administered a PE/PET polymer fabric for a minimum of 13 weeks at target concentrations of 0.5%, 2.5%, and 5%. Milling the fabric under cryogenic conditions was necessary to obtain a fine powder suitable for the feeding study. Once milled and mixed with feed, the presence of antimony in the PET provided a convenient marker for the presence of the test material in the feed. Analysis for antimony in feces and blood was also done to assess if absorption of antimony occurred.

Antimony was found in the blood of all 20 high-dose group animals, indicating that, under the conditions of exposure, it was bioavailable from the milled polymer fabric in the diet. This observation was of interest, because no antimony was detected in extraction tests of the intact polymer fabric with simulated saliva and gastric fluid (data not shown). This suggests that the greatly increased surface area of the finely ground polymer material and/or actual conditions in gastrointestinal tract may have played a role in the bioavailability of the antimony. The typical structure of the polymer fabric (i.e., not milled) presumably further limits possible exposure to antimony from the polymer fabric if used for a product held in the mouth or if it would be ingested.

None of the end points evaluated in the current polymer fabric feeding study suggested relevant toxicity due to the test article despite the presence of a substantial potion of polymer in the diet and apparent absorption of antimony. Previous investigations of antimony trioxide have indicated an inconsistent effect on red blood cell counts. Sungawa reported a decrease in red blood cell count in rats given 418 mg antimony trioxide/kg body weight (bw)/day for 24 weeks, whereas Smyth and Thompson reported an increased red blood cell count in rats treated with same compound at a dose of 894 mg antimony trioxide/kg bw/day for 30 days (ATSDR 2008). Although increased red blood cell counts and hemoglobin and hematocrit levels were observed in the present study for the low- and mid-level groups, we do not consider these findings to be of toxicological relevance given the lack of a dose-response in the findings (effects absent at the high level) as well as the fact that these clinical parameters were well within historical values for this strain of rat.

The lack of any toxicological effects attributable to antimony exposure in the present study is not surprising given the results of a report by Hext, Pinto, and Rimmel (1999). In that study, Wistar-derived rats (Alpk:ApfSD) were treated with antimony trioxide via the diet at concentrations of 0, 1000, 5000, or 20000 ppm for 90 days. Following treatment, some minor changes in urine and plasma biochemistry and hematology (including slightly increased red blood cell counts in the high-dose group males, but not females) were observed. In addition, liver weights were increased slightly in both males and females at the high dose level, but without corresponding histological effects. None of these changes were considered by the authors to be of toxicological significance, and the concentration of 20000 ppm in diet, equivalent to about 1686 mg of antimony trioxide per kg of body weight per day was considered to be a no observed adverse effect level. Based on body weight and food consumption data from the present study, we estimate an antimony intake of about 0.5 to 0.7 mg/kg bw/day. Thus, the level of antimony exposure achieved in the current feeding study involving the administration of the polymer fabric, which contains about 130 ppm antimony trioxide, was far less than the no observed adverse effect established by Hext, Pinto, and Rimmel (1999).

In the Salmonella reverse mutation study, the finely ground polymer material was extracted in PBS and DMSO. These extracts were evaluated over a range of concentration for mutagenic potential in TA98, TA100, TA102, TA1535, and TA1537 tester strains of Salmonella typhimurium both with and without metabolic activation with Aroclor 1254–induced rat liver post mitochondrial S9 fraction. During preliminary dose range–finding testing, a partial positive dose-response was observed in the PBS extract, nonactivated TA98 test. However, the results of the confirmatory test showed no indication of a mutagenic response. No other potential positive mutagenic responses were observed, and neither the PBS nor the DMSO extracts were found to be cytotoxic to bacteria at any of the dose levels tested.

The results of these studies demonstrate that repeated daily ingestion of a spunbond, nonwoven polymer fabric consisting of polyethylene and polyethylene terephthalate for a period of up to 13 weeks is well tolerated in rats, with no apparent target-organ toxicity at dietary levels up to 5%, and that extracts of this fabric are not mutagenic in a bacterial reverse mutation assay.